The present invention is directed to nucleic acid and protein sequences that encode a cysteine-containing tag and a targeting protein. The present invention is also directed to a biological conjugate comprising proteins having the above protein sequences and a binding moiety covalently bound to the cysteine-containing tag in the protein. The present invention is also directed to pharmaceutical compositions containing the biological conjugates in combination with selected therapeutic, diagnostic, or research entities, and methods of administering the pharmaceutical compositions to a patient to achieve targeted delivery of the therapeutic, diagnostic, or research entities in the patient. 2. Description of the Related Art.
Conjugation of recombinant proteins to various entities is used in several areas. One area is targeted delivery of therapeutic, diagnostic, and research agents to targeted cells in the patient in order to improve their efficacy and to minimize potentially adverse side effects. In this area either therapeutic, diagnostic, and research agents, or their carriers are chemically conjugated to recombinant targeting proteins that can selectively bind to targeted cells (reviewed by Dubowchik & Walker, 1999). The resulting conjugates are structurally and functionally heterogeneous because they are formed randomly via chemical reactions with few of several available chemical groups, usually ε-amino groups of lysine residues, in the targeting protein. Since random conjugation does not discriminate between functionally important and dispensable amino acid residues in the targeting protein, the procedure should be custom-developed and optimized on a case-by-case basis in order to increase the proportion of functionally active proteins.
Another area is derivatizing artificial surfaces and/or bulk compositions of biomedical devices or tissue scaffolds with proteins that target certain components of intra-organism environment in order to improve surface compatibility with the environment and to modulate the desired features, such as affinity or rejection of certain intra-organism components. In this area recombinant proteins are covalently grafted on the material through random chemical conjugation, usually via ε-amino groups of lysine residues, that involves both functionally important and dispensable amino acid residues in the proteins, resulting in heterogeneous products with unknown fraction of functionally active proteins.
Yet another area with similar problems is construction of various biosensors or other functional devices with protein-derivatized surfaces that convert the results of interactions between the “working” protein and the targeted components of the environment into a detectable output, including but not limited to a detectable signal or the products of enzymatic activity of immobilized proteins. In this area recombinant proteins are also chemically conjugated to artificial surfaces of these devices usually via ε-amino groups of lysine residues, yielding heterogeneous surfaces with unknown fraction of functionally active proteins.
Several methods for chemical conjugation of proteins to artificial surfaces have been developed (see, for example U.S. Pat. No. 5,492,840 to Malmqvuist; U.S. Pat. No. 5,853,744 to Mooradian et al.; U.S. Pat. No. 6,033,719 to Keogh, Mann et al. (2001); Kuhl & Griffith-Cima, (1996); Bentz et al. (1998). These methods were developed on a case-by-case basis in order to minimize damage to the protein and to increase the homogeneity of the surface.
These problems are well recognized, and over the years several approaches have been developed for introduction into recombinant proteins of unique cysteine residues for site-specific conjugation of various entities. This strategy is based on observation that intrinsic cysteine residues in proteins are usually involved in intramolecular or inter-subunit disulfide bonds and are not readily available for chemical conjugation. In theory, introduction of a unique cysteine residue that does not affect formation of intrinsic disulfide bonds and does not affect functional activity of the recombinant protein can provide a thiol group available for site-specific conjugation via chemistries known in art. For example, several groups reported introduction of cysteines into recombinant single-chain Fv antibody fragments (scFv), usually at or near C-termini, in order to use these cysteine residues for formation of diabodies and/or for site-specific conjugation to various entities (Adams et al., 1993; Kipriyanov et al., 1994; Wang et al., 1997; Marti et al., 2001; Gupta et al., 2001; Xu et al., 2002; Li et al., 2002; Renard et al., 2002; Albrecht et al., 2004). However, even for scFv, the presence of unpaired cysteine at or near the C-terminus significantly affects protein yield, solubility and functional activity (Schmiedl et al., 2000). Futami et al. (2000) introduced cysteine residues near the N- and C-termini of into human RNase I which resulted in a stabilized RNase I. However, yield and enzymatic activity of the product were significantly reduced. Moreover, this mutant RNase I or its fragments were not used in other products.
Another method for site directed modification of proteins is intein-mediated ligation of various entities to the C-terminus of the protein (see, for example Evans et al., 1999; Tolbert and Wong 2000; Macmillan et al., 2000; Mukhopadhyay et al., 2001; Hofmann, and Muir, 2002; Lovrinovic et al., 2003; Wood et al., 2004). However application of this method require proper folding of the protein fused to a large intein domain and the ability to withstand fairly harsh reducing conditions during intein-mediated ligation. Furthermore, in both approaches discussed above, conjugation to available cysteine residue is limited to entities that do not interfere with activities of the protein despite their close proximity to the body of the protein.
Thus, in the area of protein-based targeted delivery of therapeutic, diagnostic, and research compounds, as well as in the area of construction of various devices and scaffolds with protein-derivatized surfaces, what is needed in the art are compositions and general methods that (1) allow for site-specific conjugation of recombinant proteins to various entities in order to produce more homogeneous products in ways that minimize interference with functional activities of said proteins; (2) readily convert various recombinant proteins of interest into a format suitable for site-specific conjugation; (3) can be utilized with a wide variety of entities to which a recombinant protein of interest need to be conjugated; and (4) do not result in immunogenic or toxicity problems when introduced into humans. The present invention is believed to be an answer to these objectives.